1. Field of the Invention
The present invention relates to a method for producing a DNA chip (DNA microarray) in which several thousand to not less than ten thousand different types of DNA fragments are aligned and fixed as minute spots at a high density on a base plate such as a glass microscope slide.
2. Description of the Related Art
Methods for analyzing genetic structure have been remarkably progressed in recent years. A large number of genetic structures represented by those of human genes have been clarified. The analysis of genetic structures as described above uses a DNA chip (DNA microarray) in which several thousand to not less than ten thousand different types of DNA fragments are aligned and fixed as minute spots on a base plate such as a glass microscope slide.
In general, the DNA chip is produced by arranging a plurality of minute spots of a sample solution containing DNA fragments on a base plate such as glass. Those widely used as a method for forming the minute spots are based on a system such as the QUILL system, the pin and ring system, and the solid pin system in which a sample solution containing DNA fragments is supplied (stamped) onto the base plate by using a so-called pin. Even when any one of the foregoing methods is adopted, it is necessary to suppress the dispersion of the volume and the shape of each of the minute spots to be low so that the distance between the respective minute spots is maintained to be constant.
The PCR amplification step is used to prepare the sample solution containing the DNA fragments. The sample solution is often used while performing the amplification up to an amount of liquid required for the spot starting from a slight amount of original DNA. The amount of liquid obtained by the amplification is about several tens of microliters, and the reagents required for the amplification are expensive. Therefore, it is desirable to conserve reagents, which yields a more efficient use of the obtained liquid.
On the other hand, it is also desirable to realize a higher spot density. In this regard, it is necessary to develop a new method in which the shape control performance is satisfactory for the minute spot, and excellent productivity is realized as well.
When the minute spots are formed on the base plate by supplying the sample solution, the sample solution is prepared by PCR-amplifying a DNA fragment in a preparation vessel such as a cartridge beforehand to prepare a PCR product, drying the obtained PCR product to give DNA powder, and dissolving the obtained DNA powder in a buffer solution.
The sample solution is charged in a supply apparatus. The sample solution is supplied onto the base plate by using the supply apparatus to form the minute spots on the base plate.
In this procedure, the step of preparing the sample solution and the step of supplying the sample solution are separate from each other. Therefore, it is necessary to additionally perform management between the steps, and it is required to provide an equipment for preserving the sample solution. Further, the sample solution more probably contacts with the atmospheric air, and hence it is feared that the quality of the sample solution is deteriorated.
Further, the following problem arises because the sample solution is prepared in the preparation vessel such as the cartridge. That is, when the sample solution after the preparation is transferred to a pipette, a part of the sample solution remains in the cartridge. Further, when the sample solution is supplied to the supply apparatus by the aid of a pipette, a part of the sample solution also remains in the pipette. This procedure is also disadvantageous in the efficiency of utilization of the sample solution.
The present invention has been made taking the foregoing problems into consideration, an object of which is to provide a method for producing a DNA chip, which makes it possible to perform a series of steps of the preparation of a sample solution to the supply process without deteriorating the quality of the sample solution, improve the efficiency of utilization of the sample solution, realize the simplification of the preservation equipment for the sample solution, realize inexpensive cost, and improve the quality of the DNA chip.
According to the present invention, there is provided a method for producing a DNA chip by supplying a large number of sample solutions onto a base plate, comprising the steps of PCR-amplifying a DNA fragment to prepare a PCR product; drying the PCR product to prepare DNA powder; supplying the DNA powder into a solution supply apparatus; and supplying a buffer solution into the supply apparatus to prepare a sample solution; wherein the sample solution in the supply apparatus is supplied onto the base plate by using the supply apparatus to produce the DNA chip.
That is, in the present invention, the process for mixing the DNA powder and the buffer solution to prepare the sample solution, and the step of supplying the sample solution onto the base plate are performed in the identical supply apparatus. By doing so, the sample solution in the preparation vessel is moved in a powder state into the supply apparatus. Accordingly, it is possible to reduce any sample residue adhered, for example, to the vessel wall in the preparation vessel. Further, for example, it is unnecessary to use any pipette to move or transfer the sample. Thus, it is possible to avoid the occurrence of any residue of the sample remained and discarded in the pipette.
According to another aspect of the present invention, there is provided a method for producing a DNA chip, comprising the steps of PCR-amplifying a DNA fragment to prepare a PCR product; supplying the prepared PCR product into a solution supply apparatus; drying the PCR product in the supply apparatus to prepare DNA powder; and supplying a buffer solution into the supply apparatus to prepare a sample solution; wherein the sample solution in the supply apparatus is supplied onto the base plate by using the supply apparatus to produce the DNA chip.
That is, in the present invention, the process for drying the PCR product to prepare the DNA powder, and the process for mixing the DNA powder and the buffer solution to prepare the sample solution are performed in the identical supply apparatus.
Accordingly, it is possible to reduce the loss which would be otherwise caused, for example, by any scattering of the sample in the drying step. Thus, it is possible to improve the efficiency of utilization of the sample solution. Further, the series of operations, i.e., from the preparation of the DNA powder to the supply process, are performed in one supply apparatus. Therefore, the sample solution scarcely contacts with the atmospheric air. Thus, it is possible to avoid any deterioration of the quality of the sample solution.
According to still another aspect of the present invention, there is provided a method for producing a DNA chip, comprising the steps of PCR-amplifying a DNA fragment to prepare a PCR product in a solution supply apparatus; drying the PCR product in the supply apparatus to prepare DNA powder; and supplying a buffer solution into the supply apparatus to prepare a sample solution; wherein the sample solution in the supply apparatus is supplied onto the base plate by using the supply apparatus to produce the DNA chip.
That is, in the present invention, the series of steps ranging from the PCR amplification to the supply process are performed in the identical supply apparatus. Accordingly, the steps from the preparation of the sample solution to the supply process can be performed in accordance with the series of steps without deteriorating the quality of the sample solution. Further, it is possible to realize the simplification of the preservation equipment for the sample solution. It is possible to reduce the cost and improve the quality of the DNA chip.
It is unnecessary to perform any step of transferring the sample solution to another vessel. Therefore, it is possible to further improve the efficiency of utilization of the sample solution. Further, the steps ranging from the amplification of DNA to the supply process are performed on one supply apparatus. Therefore, the sample solution scarcely contacts with the atmospheric air, and thus it is possible to avoid any deterioration of the quality of the sample solution.
According to still another aspect of the present invention, there is provided a method for producing a DNA chip, comprising the step of PCR-amplifying a DNA fragment to prepare a PCR product in a solution supply apparatus; wherein the sample solution after preparation in the supply apparatus is supplied onto the base plate by using the supply apparatus to produce the DNA chip.
That is, the PCR product, which is obtained by the PCR amplification in the supply apparatus, is directly supplied onto the base plate.
By doing so, in addition to the respective functions and effects of the inventions described above, the step of preparing the sample solution in the vessel is simplified. It is possible to efficiently produce the DNA chip in a short period of time. It is also preferable to pour a reagent to neutralize the action of any component that inhibits the hybridization action on the DNA chip, that is included in any reagent used during the amplification, or exists in the solution containing the PCR product in the supply apparatus.
It is preferable that the sample solution is supplied in accordance with an ink-jet system. In this case, it is preferable that the supply apparatus is a dispenser comprising a plurality of arranged micropipettes each including a pouring port for pouring the sample solution from the outside, a cavity for pouring and charging the sample solution thereinto, and a discharge port for discharging the sample solution, formed on at least one or more substrates, the micropipette further including a piezoelectric/electrostrictive element disposed on at least one wall surface of the substrate which forms the cavity so that the sample solution is movable in the cavity, and mutually different types of the sample solutions being discharged from the discharge ports of the respective micropipettes.
Accordingly, the sample solution is prepared by performing the steps of pouring, for example, the DNA powder obtained by drying each of the different kinds of PCR products, each of the different kinds of PCR products, original DNA before the PCR amplification and the buffer solution to dissolve the DNA powder therein, or PCR amplification reagents (for example, primers, enzyme, PCR buffer solution, dNTP""s, distilled water) from the pouring ports into the plurality of cavities, and optionally drying the PCR product at the pouring port portion to prepare the DNA powder. After that, the piezoelectric/electrostrictive element is driven, and thus the different types of the sample solutions in the plurality of cavities can be discharged from the discharge ports to produce the DNA chip.
As described above, the supply apparatus based on the ink-jet system, in which the volume of the portion for storing the sample in the supply apparatus is about several to several tens of microliters, is suitable, for example, for the preparation of the sample solution, the amplification, the purification, and the production in the supply apparatus. The foregoing function can be simultaneously possessed, in addition to the formation of the spots onto the base plate as the original function of the supply apparatus. It is possible to produce the DNA chip extremely efficiently. Further, as described later, when the supply apparatus itself is made of ceramics which has a good thermal conductivity, for example, as compared with glass and plastics, the supply apparatus is preferred for the PCR amplification in which the thermal cycle is performed.
It is also preferable that completion of preparation of the sample solution in each of the plurality of cavities is recognized by sensing a change of a fluid characteristic in the cavity. The piezoelectric/electrostrictive element, which is formed on at least one wall surface of the substrate for forming the cavity, functions as a sensor for sensing the physical characteristic of the liquid in the cavity. Accordingly, it is possible to accurately detect the completion of preparation.
The above and other objects, features, and advantages of the present invention will become more apparent from the following description when taken in conjunction with the accompanying drawings in which a preferred embodiment of the present invention is shown by way of illustrative example.